Chlorophyll binding to Cytochrome b6f precedes photosystem I and II in barley etioplasts

Abstract

Chlorophyll (Chl) is essential for oxygenic photosynthesis, binding to membrane proteins for light harvesting and electron transfer. In angiosperms, Chl synthesis is halted in darkness, preventing accumulation of Chl-binding photosynthetic complexes in etioplasts. However, etioplasts assemble a dimeric Cytochrome b6f (Cyt b6f) complex, uniquely binding protochlorophyll (Pchl), the esterified derivative of protochlorophyllide (Pchlide). This indicates an evolutionarily conserved structural or functional role for Pchl distinct from the Chl bound in Cyt b6f in chloroplasts. Here we show that upon light-induced Chl synthesis in-vivo and in-vitro, Chl accumulation in Cyt b6f dimers precedes photosystems I and II. We find that chlorophyllide and Chl bind to the light-harvesting-like protein 3 (LIL3), supporting a role for LIL3 in early Chl allocation that extends its described role in stabilizing geranylgeranyl reductase. We determine a dissociation constant of 246.6 {+/-} 37 nM for Chlide binding to LIL3 in-vitro and show that Cyt b6f monomers and LIL3 co-migrate with Chlide in native PAGE, whereas Cyt b6f dimers and LIL3 co-migrate with Chl. These results indicate that Chlide binding to LIL3 chaperones esterification to Chl and reduction of geranylgeraniol, and that Chl release with Cyt b6f dimerization prioritizes Chl binding to Cyt b6f assembly during de-etiolation.